The following publications are considered relevant for an understanding of the invention.
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Chemokines in Health and Disease
The immune system promotes health by combating foreign pathogens, alleviating intrinsic disease and repairing physical injury. Infected, injured, or otherwise compromised tissues mobilize cells of the immune system by releasing chemo-attractants called chemokines (CKs) into the blood stream. The same CKs induce the activation of leukocytes and direct the differentiation of lymphocytes [Rossi, 2000; Zlotnik, 2000]. The deployment of immune cells is essential, not only to confront pathogenic challenge, but also for immune surveillance and tolerance to “self” [Mackay, 2001]. Close to fifty different human CKs and twenty CK receptors (CKRs) respond to multifarious pathogens and disease states. Tight regulatory control of the CK system imparts rapid, measured and apposite responses to the various pathogenic challenges. Of equal importance to health is the control which prevents the immune system from acting against “self”. An immune response which is inappropriate, excessive, or protracted, relative to the pathogenic insult, if any, will cause injury to healthy tissue. Such aberrant immune responses are responsible for the clinical conditions of multiple sclerosis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), rheumatoid arthritis, psoriasis, asthma and juvenile diabetes, the major and prevalent autoimmune diseases. Experimental studies with animal models of disease [Gerard, 2001; Dogan, 2004; Szekanecz, 2003] and clinical observations [Gerard, 2001; Godessart, 2001] indicated that the levels of CKs and their cognate receptors correlate with specific autoimmune diseases.
Current Treatment of Autoimmune Disease
The etiology of no single autoimmune disease is known, but disease pathology, in every case, is the result of immune cell activity directed against “self”. It follows, therefore, that the “state of the art” treatment is restricted to the induction and maintenance of disease remission. Multiple sclerosis (MS), the most common cause of neurological disability in young adults, presents with a relapsing-remitting course and is followed by a secondary progressive phase. Steroids are the first choice of treatment to shorten the duration of relapse and accelerate recovery. Long term treatment, with Non Steroidal Anti Inflammatory Drugs (NSAIDs), is given to maintain, or extend remission. Inflammatory bowel disease (IBD) affects over 1 million Americans today. Manifested by severe abdominal pain and diarrhea, it is associated with an increased risk of bowel cancer. For patients suffering mild IBD, remission is induced with NSAIDs. Because of the adverse side effects of steroids, they are reserved for patients with moderate to severe disease and for those who do not respond to NSAIDs. When the disease is refractory to steroids, immunosuppressant drugs are used.
Established and new treatments of autoimmunity act by modulating and if necessary, suppressing the immune system. Both approaches run the risk of side effects, the short term of which are known, the long term, which cannot always be predicted. The shortcomings of steroids, the first line of treatment for severe MS and IBD are well documented: gastrointestinal, dermatologic, neurological, endocrinological, ophthalmic and metabolic side effects. Remission in MS is “extended” by interferon beta and glatiramer acetate (Copaxone), but drug efficacy, tantamount to a 30% reduction in the frequency of relapse, is offset by severe side effects, such as fatigue, pain and bladder dysfunction. The range of medications extending remission in IBD is wider, but the side effects no less severe. NSIADs are intolerable to a significant number of IBD patients and the alternatives, the immuno-suppressants 5-AZT and 6-mp, cause severe side effects. Prolonged use of a new generation of antibody drugs, exemplified by Remicade, is beginning to reveal a negative side. Remicade has been linked with tuberculosis, opportunistic infection and the activation of latent MS (Centocor, Inc. 2005). Add to this deteriorating safety profile the high cost and administration by intravenous infusion and this innovative treatment becomes a less than an attractive alternative to tried and tested medications. There is a need for improved medication to treat acute inflammation in disease relapse and provide long term treatment to maintain remission.
Chemokine Receptors (CKRs) as Conventional Drug Targets
The CKR, a member of the G-protein Coupled Receptor (GPCR) family, has a drug target pedigree. More than forty five percent of all marketed drugs target GPCRs [Horuk, 2003]. Given that the ligands of GPCRs are low molecular weight peptides (histamine, dopamine, serotonin), it was predicted that CKRs would be tractable targets for small molecule drugs (Proudfoot et al., 2003). The majority of drug candidates targeting CKRs, including those under development and those which have been abandoned, are indeed small molecule inhibitors [Wells, 2006]. Based on steric and energetic considerations [Onuffer, 2002] and empirical studies [Sabroe, 2000], it was proposed that small molecule antagonists of CKRs should be non-competitive inhibitors of CKs that block ligand-receptor interaction by stabilizing the receptor in an inactive conformation.
CKRs are known to interact with both their cognate and with unrelated CK ligands, a phenomenon known as “redundancy”. As a functional definition of CK—CKR interaction, a “cognate” CKR—CK pair is defined as a CKR and a CK for which the CKR is activated by nanograms of the CK to induce intracellular signaling (calcium mobilization, kinase and lipase activation). With respect to the same CK—CKR system, an “unrelated” CKR—CK combination is defined as a CKR which requires at least three orders of magnitude more of CK, micrograms, to elicit a positive, negative, or unproductive intracellular response.
The identity of CKRs as GPCRs is an evident advantage for drug development. The atypical involvement of these GPCRs in physiological immunity, however, makes CKRs equivocal drug targets and even a therapeutic liability. Antibodies against the receptor, CCR2, which is expressed by inflammatory T cells and monocytes, were used to treat experimental collagen-induced arthritis [Bruhl, 2004]. The treatment was therapeutic during disease initiation (day 0-15), but deleterious in disease progression (day 21-36). It transpired that a sub-population of regulatory T cells, responsible for immune tolerance, is expanded several fold during the phase of disease progression. The antibodies, by blocking the CCR2 receptors of regulatory T cells, exacerbated disease symptoms. Inhibition of the same drug target expressed by different cell types, CCR2 in the example, can be therapeutic, or pathologic, depending on the function of the cell expressing the targeted CKR. The CK, RANTES/CCL5* (*CK classification: [Zlotnik, 2000]) and its cognate receptor, CCR5, are examples of disease-related proteins that are also essential for physiological immunity. Elevated levels of CCL5 and CCR5 correlated with glomerular cell proliferation and macrophage infiltration in experimental glomerulonephritis. CK analogues (Met—RANTES and amino-oxypentane—RANTES), which block the receptor, reduced glomerular cell proliferation and macrophage infiltration, but aggravated clinical symptoms [Anders, 2003]. The CK analogues were observed to be therapeutic antagonists of leukocyte recruitment, but pathogenic activators of resident macrophages. CKRs, validated GPCR drug targets, are potential therapeutic liabilities as mediators of physiological immunity.
CKs and CKRs constitute a network of interacting proteins [Schwarz, 2002]. Drugs directed at individual proteins of a network risk perturbing the network as a whole. It follows, therefore, that when interacting and interdependent proteins of the network are essential for physiological immunity, targeting one of the proteins may affect the overall function of the immune system and so reduce the drug's efficacy. CK gene knock-out has been the preferred experimental approach to study the physiological consequences of CK network interference. In a comparative study of inflammatory cells from MCP1/CCL2 null mice and their wild type counterparts, it was found that eliminating MCP-1/CCL2 altered immune responses to disease [Ferreira, 2005]. Equivalent tissues and cells from null and wild-type diseased mice showed significant differences in their respective CK and CK receptor profiles. Network principles were also observed in tissues derived from the MCP-1/CCL2 null mice. Reconstituted expression of MCP-1/CCL2 in macrophages of the CK knock-out mice selectively suppressed the expression of CKs, those specifically induced by MCP-1/CCL2 inactivation. Physiological immunity in genetically altered animals, as in their wild type counterparts; was dictated by principles of network responses and adaptation.
In experimental animal models of disease, the relevance of networking is most conspicuous at the cellular level. Experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis, was used to study the role of CCR2 in disease development [Gaupp, 2003]. The recruitment of monocytes and macrophages into the CNS was examined in CCR2 knock-out, disease-induced mice. Contrary to what was expected, inactivation of CCR2 did not confer resistance to EAE in the mice. CNS lesions did contain diminished levels of monocytes, proof of impaired monocyte function, but also elevated levels of neutrophils. CK (IL-8/CXCL8) and CK receptor (CCR1, CCR5) profiles of the experimental mice were found to be consistent with augmented neutrophil levels in the lesions. It was proposed that the CK—CKR network of the CCR2 null mice responded to disease induction with a “compensatory” immune response involving alternative immune regulator molecules and effector cells. The foregoing example is yet another expression of the network principle. Neutralization of a disease-related protein, one which is important for physiological processes in general, can elicit a counter response, which itself can be pathological. A murine model of experimental arthritis, which reproduces the symptoms of severe human rheumatoid arthritis, provided another illustration of the physiological consequences of network interference [Quinones, 2004]. CCR2 knock-out mice were used to study the role of the receptor in experimental arthritis. The CKR is implicated in disease pathogenesis, therefore, it was not anticipated that the phenotype of the CCR2 knock-out mice would be similar to that found in severe human arthritis, elevated T cell levels and monocytes and macrophages concentrated in the inflamed joints. The proffered explanation was that disease in the CKR compromised mice stimulated the expression of alternative CKRs to mobilize the inflammatory cells. The consequence of perturbing a physiologically essential network, even for therapeutic effect, can be to elicit compensatory reactions to consolidate, but potentially exacerbate, the status quo.
The CK—CKR axis, despite the “druggable” attributes of GPCRs and their small molecule ligands, is demonstrably problematic as a drug target. It functions as a network of interactive and interdependent regulatory proteins central to physiological immunity. Compensatory and possibly deleterious responses may therefore be the unavoidable consequences of pharmacological intervention in the network.
The Chemokine—Chemokine Receptor Network
A disproportionate number of chemokine (CK) ligands, ill-defined ligand specificity and equivocal CK functionality in vitro, were cited as proof of CK “redundancy” and worse still, “promiscuity”. Such epithets, although consistent with data at the time, were incompatible with a CK—Chemokine Receptor (CKR) network essential for physiological immunity and pathological autoimmunity. Re-examination of the original data, in the light of subsequent functional studies and structure-activity analyses, reveals physiological relevance in both the ligand to receptor ratio and ostensibly indiscriminate receptor activation. To begin with, CKs, which are agonists of their cognate receptors, were found to be natural antagonists of unrelated receptors, as determined by chemotaxis and Ca2+ flux assays. Three agonists of the receptor, CXCR3, MIG/CXCL9, IP10/CXCL10 and I-TAC/CXCL-11, were shown to be antagonists of the unrelated receptor, CCR3 [Loetscher, 2001]. The most potent of the three, I-TAC/CXCL11, was found to be a natural antagonist of CCR5 [Petkovic, 2004]. Eotaxin/CCL11, an agonist of CCR3, was also shown to be an agonist and natural antagonist of the unrelated receptors CCR5 and CCR2, respectively [Ogilvie, 2001]. Another cognate ligand and agonist of CCR3, Eotaxin3/CCL26, was found to be a natural antagonist of unrelated receptors CCR1 and CCR5 [Petkovic, 2004]. MCP3/CCL7, a cognate agonist of receptors CCR1, 2 and 3, was shown to be a natural antagonist of the receptor, CCR5 [Blanpain, 1999]. The concomitant activation and inhibition of CKRs was interpreted to be a mechanism for regulating the recruitment of functionally discrete sub-populations of leukocytes. To illustrate the point, MIG/CXCL9 and Eotaxin/CCL11, by altering the balance between Th1 and Th2 cells, could determine the course of Allergic Airway Inflammation [Thomas, 2004; Fulkerson, 2004]. MIG/CXCL9 is an agonist of CXCR3, expressed by Th1 cells and an antagonist of CCR3, expressed by Th2 cells. Eotaxin/CCL11, a cognate ligand of CCR3, was also shown to have a high affinity binding site in the unrelated receptor, CXCR3 [Xanthou, 2003]. A refinement of the point, activation/inhibition of CKRs, was regulation of T cell recruitment by differential activation of the same receptor [Thomas, 2002]. IP-10/CXCL10 and MIG/CXCL9, cognate ligands of CXCR3, could respectively enhance, or diminish eosinophil accumulation and airway hyper-reactivity. Cumulative data now show that the disease-related distribution and activities of immune cells are in fact a corollary of CK multiplicity and specificity for diverse CKRs.
Since neither experimental data, nor clinical evidence distinguishes between wild type and disease-related CKs, it is presumably the same chemo-attractants that mobilize the immune cells of physiological and pathological immune responses. CK pathogenicity appears to be the consequence of inordinate, inappropriate and enduring wild-type CK activity, phenomena consistent with CK network deregulation. CK to receptor binding analyses were carried out to elucidate the molecular bases for the regulation of CK—CKR interactions. MIG/CXCL9 and IP-10/CXCL10 bind competitively the receptor CXCR3 but non-competitively with respect to the third related CK, ITAC/CXCL11 [Cox, 2001]. In this example, functionally related CKs were shown to interact with discrete and overlapping sites in their cognate receptor. The anomalous activities of CKs, those of CKs interacting with unrelated receptors, may be more informative about CK network regulation per se. For example, the cognate ligands of CXCR3 inhibited CCR3 functional responses and were observed to displace that receptor's cognate ligand, Eotaxin/CCL11 [Loetscher, 2001]. A more detailed study of the same system disclosed that ITAC/CXCL11, cognate ligand of CXCR3, efficiently displaced Eotaxin/CCL11 from the extra-cellular loops of the latter's receptor, CCR3. Cognate and unrelated ligands, therefore, can share overlapping binding sites within the same receptor [Xanthou, 2003]. Another anomalous, but informative observation, concerns Eotaxin/CCL11 and its unrelated receptor, CXCR3. Although Eotaxin/CCL11 is neither an agonist, nor an antagonist of CXCR3 in vitro, the receptor has a high-affinity binding site for this CK which can be competitively occupied by ITAC/CXCL11 [Xanthou, 2003].
Chemokines as Innovative Drug Targets
An upshot of the molecular structure-activity analyses and binding studies is that receptors comprise CK binding sequences that, given the context, are regulators of CK activity. In the context of the CKR, regulatory elements are potential, but problematic drug targets, because the receptors are essential for physiological immunity.
Functional studies and complementary molecular analyses of CK receptors, have disclosed regulatory sequences in the receptors of physiological and disease-related CKs. Anomalous interactions of CKs with unrelated receptors implicate ubiquitous cryptic regulatory sequences important for general CK—CKR network activity. CKR derived CBPs, capable of modifying CK binding activities, are not without precedent. In a study to identify and define receptor sequences responsible for IL-8/CXCL8 and GRO-α/CXCL1 binding, receptor-derived sequences were shown to be competitive inhibitors of CK binding [Gayle, 1993]. A peptide derived from an extra-cellular domain of the same receptor, CXCR1, was found to be an antagonist of the cognate CK, IL-8/CXCL8 [Attwood, 1996] and when chemically modified, the peptide was made a stronger inhibitor [Attwood, 1997]. More recently, an MCP1/CCL2 binding peptide, homologous to a sequence in an extra-cellular loop of the CKRs CCR2 and CCR3, was shown to be angiostatic by antagonizing MCP-1/CCL2 binding to CCR2 [Kim, 2005].
Mechanism of Drug Action—Redress of Immune Imbalance
The pathogenesis of all autoimmune diseases is patently de-regulated and dysfunctional immune activity. A challenge for any therapeutic approach is to manipulate immune processes that, in the same individual, both cause clinical symptoms and protect against disease. To date, no therapy has met the challenge satisfactorily. Autoimmune diseases are treated with low efficacy drugs which cause significant side effects. Most of the current drugs are small synthetic molecules whose deficiencies appear to derive from their mechanism of action, irreversible inhibition of disease related proteins.
Drug candidates targeting CKRs belong to the class of small synthetic molecules [Wells, 2006]. Following from the preceding discussion, drugs of this type are inherently flawed. None can be expected to block and neutralize a CKR with impunity, given the organization of CKs and their receptors as a network which is essential for physiological immunity. The principles of network activity govern immune responses at the molecular and cellular levels and dictate the outcome, therapeutic, or deleterious, of inhibitory drug activity. The overriding objective of the network is to maintain immune balance, any perturbation of the network eliciting a response to redress the balance. Experimental models of autoimmune disease and clinical evidence provide manifold examples of network activity where disease manifests itself as immune imbalance and health is restored by redressing the balance. Take, for example, allergen challenge in the lung, which elicits deleterious eosinophils and protective Th1 lymphocytes [Fulkerson, 2004; Thomas, 2002]. Allergen-induced CK, Eotaxin/CCL11, recruits inflammatory eosinophils. At the same time, Th1 cells express the CK, MIG/CXCL9, a natural inhibitor of eosinophils. The inference was that the inflammatory status of the allergic lung was dictated by competition between positive and negative regulatory CKs, the result of which competition was translated into a balance between Th1 and eosinophilic cells. The clinical symptoms of type 1 diabetes, herpes stromal keratitis and multiple sclerosis, are infection, tipping of the T helper (Th) lymphocyte balance in favor of a type 1 milieu [Christen, 2004]. Support for the hypothesis was provided by the diabetogenic RIP-LCMV mouse model. Type 1 CKs and cytokines were shown to be responsible for the diabetic state of the RIP-LCMV mouse. It was suggested, with qualification, that redressing the immune imbalance by inhibiting the type 1 inducing factors, or administering Type 2 cytokines, could be therapeutic. Immune balance is argued to be a salient determinant of disease progression in relapsing-remitting multiple sclerosis [Nakajima, 2004]. Levels of Th1-related CKs are elevated in the active phase of MS whereas MCP-1/CCL2, reported to induce Th2 reactions, is elevated in remission. An analysis of blood from patients with active MS revealed elevated levels of Th1 lymphocytes and significantly reduced levels of monocytes expressing CCR2, the cognate receptor of MCP-1/CCL2. The results were interpreted as evidence that elevated MCP-1/CCL2 and a subset of peripheral monocytes expressing CCR2, may correct the Th1/Th2 imbalance to create conditions for disease remission.
The Th1-Th2 dichotomy influences immune balance in physiological immunity and is a determining factor in causing the immune imbalance that is characteristic of autoimmune disease. Under prevailing physiological conditions, Th1 and Th2-like cells are in equilibrium, contributing to the establishment and maintenance of immune balance. Innate and extraneous stimuli induce Th1 and Th2-like immune responses that create new, transient equilibria and immune imbalance. Responses that entail modification of the immune cell repertoire establish a new Th1-Th2 equilibrium, restoring immune balance. In autoimmune disease, a loss of tolerance is expressed as pathological Th1-Th2 disequilibrium and immune imbalance. In the absence of treatment, resolution of the Th1-Th2 disequilibrium is transient and an enduring immune imbalance leads to relapse. Effective treatment of an autoimmune disease, therefore, must restore Th1-Th2 cell equilibrium to redress the immune imbalance for tolerance and disease remission. It follows, therefore, that one way to treat autoimmune disease is to alter the pathogenic equilibrium state. A pharmacological intervention is required to create a new equilibrium and to redress immune imbalance for tolerance. Its application must be incremental and in keeping with the principles of equilibrium dynamics for maximal therapeutic effect and minimal, detrimental side effects.
A recurrent theme in the study of autoimmune disease is the intercalation of physiological immunity with pathological autoimmunity. Evidence has been presented to show that the former state is achieved when immune regulators and effectors are in balance and the latter, disease state, when they are in imbalance. Given that the balance is contingent on a network of regulatory proteins, negation of a protein, albeit disease related, portends imbalance. Efficacy, therefore, must be sought in a drug which modulates and does not negate the disease-related activity of the network-associated protein.